Understanding the molecular mechanisms underlying the human ?- to ?-globin gene switch has long been recognized as important in the treatment of hemoglobinopathies such as sickle cell disease (SCD), Cooley's anemia and ?-thalassemias, since a wealth of evidence demonstrates that increased fetal hemoglobin (HbF) significantly decreases the pathophysiology associated with these diseases in patients. Our goal in this study is to understand the role of post-translation modifications (PTMs) of transcription factors involved in ?-globin gene silencing during the adult definitive erythropoiesis. Our clinical goal is to identify new molecular targets based on the outcome of this study that can be modulated therapeutically for up-regulation of ?-globin synthesis to treat these diseases. We recently demonstrated that one mode of ?-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the A?-globin or G?-globin gene CAP sites, respectively, and is mediated through the DNA-binding moiety GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi2, a component of the NuRD chromatin remodeling complex. Post-translational modifications of transcription factors may play a critical role in regulating the formation of transcriptional repressor complexes or activator complexes, particularly when both types of complexes might utilize the same DNA-binding protein, as is the case for GATA-1 in erythroid cells. We propose that the O-GlcNAcylation (O-GlcNAc) PTM is involved in regulating ?-globin transcription by controlling assembly of the GATA-1-FOG-1-Mi2 (NuRD) transcriptional repressor complex upstream of the ?-globin genes. A single Specific Aim will test the hypothesis that O-GlcNAcylation of GATA-1-FOG-1-Mi2 (NuRD) transcription/chromatin remodeling factors regulates their participation in a multi-protein repressor complex. In Specific Aim 1a, we will investigate the regulation of the ?-globin genes by O-GlcNAcylation. Chromatin immunoprecipitation (ChIP) analyses will be utilized to examine the spatial and temporal arrangement of the O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), the GATA-1-FOG-1-Mi2 axis of proteins at the A?- globin promoter and the O-GlcNAc status of the promoter itself in cell cultures following terminal differentiation or induction of globin gene expression, with or without OGT/OGA knockdown or OGA inhibition, or in ?-YAC transgenic mice during fetal liver definitive erythropoiesis. In Specific Aim 1b, we will determine whether O-GlcNAc regulates ?-like globin gene expression during erythropoietic development in vivo in erythroid-specific floxed OGT conditional knockout ?-YAC mice, or in OGT or OGA enforced-expression ?-YAC bi-transgenic (bigenic) mice. Our proposed studies represent a new avenue of research in the globin gene switching field. The knowledge we gain from these studies will reveal novel therapeutic targets for which highly-specific treatments may be developed to increase HbF for the treatment of these red blood cells diseases without the side-effects associated with broad-spectrum therapies.